Cell division modulates prion accumulation in cultured cells

Ghaemmaghami et al. 10.1073/pnas.0708372104.

Supporting Information

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Fig. 5. PrP^Sc levels in ScN2a cell cultures can be measured quantitatively by capture ELISA. (A) Sandwich ELISA protocol. ScN2a cells were lysed and the extract digested by PK. PK-resistant proteins were denatured by the addition of 4 M GdHCl and subsequently refolded by 10-fold dilution in PBS buffer. The refolded PK-resistant proteins were captured and detected by anti-PrP antibodies, as described in Materials and Methods. (B) The limit of detection for the sandwich ELISA protocol was »0.5 ng. Different concentrations of recombinant MoPrP(89-230) were prepared in PBS containing 1% BSA and quantified using the capture ELISA protocol. The raw absorbance readout is plotted as a function of total amount of MoPrP(89-230). The error bars represent the standard deviation of three replicate measurements. (C) The sandwich ELISA protocol can quantitatively analyze changes in cellular PrP^Sc levels, and the measurements are linear within at least 1 order of magnitude. Different ratios of infected ScN2a and uninfected N2a cells were mixed before lysis. PK digestion and ELISA were conducted as described above. The raw absorbance readout is plotted as a function of the ScN2a to N2a ratio, and the error bars represent the standard deviation of three replicate measurements.

Fig. 6. (A) PrP^Sc levels in ScN2a-N2a cells cocultured at a 1:1 ratio for 35 days (open circles) compared to cultures of ScN2a (filled squares) and N2a (filled circles) cells. The relative PrP^Sc level of the coculture remains at »50% of the ScN2a culture, indicating a lack of cross-infection by exogenous PrP^Sc. (B) PrP^Sc levels in ScN2a cells treated with 1 mM QA and subsequently washed with fresh media. The x axis indicates the time after removal of QA. The relative PrP^Sc levels of the culture returns to 100% after 7 days, indicating rapid formation of PrP^Sc seeded by endogenous PrP^Sc.

SI Text

Results

Quantitative Predictions by the Limited Conversion Model.

The limited conversion model can be used to predict the fractional drop in steady-state levels of PrP^Sc induced by cell division. If we make the assumption that dividing and stationary infected cultures have the same value and has reached the same constant plateau value, then:

[7]

Thus, according to the limited conversion model, the reduction in steady-state levels of total PrP^Sc induced by cell division is the fractional ratio of and . The half-life for PrP^Sc degradation in ScN2a cells has been previously measured as »30 h (1-3), corresponding to a first-order rate constant of 0.023 h^-1. According to growth measurements in the present study (Fig. 1A), when ScN2a cells are <50% confluent, the average division time of the cells is »24 h, which corresponds to a first-order rate constant of 0.029 h^-1. Thus, according to Eq. 7, the fractional drop in PrP^Sc is predicted to be »0.44. In other words, once a steady state has been reached, the level of total PrP^Sc in dividing cells should be »44% of nondividing cells. This prediction matches well with the decrease in PrP^Sc levels observed in Fig. 2.

Solving the differential Eq. 5 provides a function for predicting changes in PrP^Sc as a function of time (t):

[8]

where is the initial and is defined by Eq. 6.

We used Eq. 8 to fit the data in Figs. 1C and 2. The rate of division changes as a function of time and decreases as the cells approach confluency. The rate of division for each discrete 1-day time interval was calculated based on the measured growth curve (Fig. 1A). We used 0.023 h^-1 as the first-order rate constant for degradation. As can be seen in Figs. 1C and 2 (solid line), the limited conversion model is able to predict both the steady-state levels and the kinetics of PrP^Sc fluctuation within the error of our experimental measurements.

Long-Term Coculture Experiment

To assess the level of infectivity transfer within wild-type cells during an extended period, we monitored the relative amount of PrP^Sc in a coculture of ScN2a and nontransfected N2a cells (1:1 ratio) compared with simultaneously grown cultures that contained either 100% ScN2a or 100% N2a cells. The relative levels of PrP^Sc in the three cultures were monitored over five weekly passages. PrP^Sc levels in the ScN2a-N2a coculture remained at »50% of that seen in ScN2a culture over the course of 35 days (Fig. 6A). These results suggest that in this N2a subline, horizontal cross-infection among a population of cells is slow relative to the rate of cell division, and PrP^Sc is primarily propagated by the transfer of infectivity from mother to daughter cells.

For comparison, we treated ScN2a cells with the known antiprion compound, quinacrine (QA), administered at its half-effective (EC₅₀) concentration, and monitored the kinetics of PrP^Sc formation after washing away the drug. In the coculture experiment (Fig. 6A), total PrP^Sc was initially reduced by 50%, because half of the culture was composed of ScN2a cells. However, with QA treatment, total PrP^Sc was initially halved, because each infected cell, on average, harbored 50% of its maximum level of PrP^Sc. In contrast to the coculture experiment, the total level of PrP^Sc fully recovered 7 days after the removal of QA (Fig. 6B). The results indicate that in ScN2a cultures, existing endogenous PrP^Sc is more effective than exogenous PrP^Sc at seeding the formation of new prions.

Methods

Coculture Experiments.

For the 7-day coculture experiment, N2a cells were transfected with an expression vector overexpressing GFP under the CMV promoter (phCMV-CGFP, Genlantis) using lipofectamine transfection reagent (Invitrogen). Stable cell lines were selected by maintaining the culture in the presence of 800 mg/ml G418 for 28 days. When observed under a microscope, »20% of the cells displayed fluorescence above background. The stably transfected cell line did not have any observable morphological phenotypes, but the cells grew at »20% slower rate compared with wild-type cells. GFP-transfected cells were mixed with ScN2a cells at a 1:1 ratio, plated on 100-mm plates at 10% confluency, and grown to confluency over the course of 7 days. Control cells containing 100% N2a-GFP and 100% ScN2a were cultured for reference. The cells were sorted using FACS Aria (Becton Dickinson). The cells were initially gated based on side and forward scatter to select for uniform cell size corresponding to the known scatter for N2a cells. Cells of uniform size were subsequently sorted based on green fluorescence as indicated in Results. A 1:1 mixture of ScN2a and N2a-GFP cells, mixed immediately before sorting, was also analyzed. Approximately 200,000 cells were sorted for each fluorescence gate. The cells were pelleted by centrifugation, washed twice with PBS, and lysed by the addition of 100 ml of lysis buffer. The PrP^Sc concentration was measured using the ELISA protocol described above.

For the 35-day coculture experiment, N2a cells were obtained by "curing" the ScN2a cells by the addition of 5 mg/ml PAMAM. We used a cured line to perform our coculture experiments with a mixture of N2a and ScN2a cells that were derived from the same subline and had exactly the same growth rate. Curing by PAMAM was achieved as described (4) and verified by PK digestion and Western blot. Cocultures of ScN2a cells and PAMAM-treated cells were plated in the appropriate ratios proportional to a 1:10 split and grown in MEM for 7 days until reaching confluency. On the seventh day, cells were dissociated and passaged, and protein lysates collected. This routine was repeated five more times over 35 days, with samples collected every seventh day. In addition, to account for PrP^Sc variability as a result of differing growth rates, seven plates each of ScN2a and PAMAM-cured cells were plated at 1:10. Protein lysate concentrations and cell counts for each of the 7 days revealed that the two cell lines had identical growth rates. Cells from confluent plates for each ratio were counted every week and showed that over the course of the 35-day experiment, the growth rate for all cell populations was equivalent. After 35 days, the collected lysates were normalized to the same protein concentration and analyzed for PrP^Sc levels using the ELISA protocol described above.

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